VLP stabilized vaccine compositions

ABSTRACT

The invention is directed to compositions and methods for the stabilization of viral and bacterial vaccines. Vaccines of the invention are contained in VLPs with stabilizing agents such as, for example, sugar alcohols (e.g., sorbitol) and degraded gelatins. Preferably the gelatin has an average molecular weight of 10,000 kilodaltons or less. These vaccines have a substantially improved thermostability as well as long term stability. The invention is also directed to the manufacture of a vaccine or the invention and methods for the administration of a vaccine of the invention to patients.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/214,526 entitled “VLP Stabilized Vaccine Compositions” filed Sep. 4,2015, the entirety of which is specifically incorporated by reference.

BACKGROUND 1. Field of the Invention

The invention is directed to compositions and methods for stabilizingbiological materials and, in particular, vaccines. In particular, theinvention is directed to vaccines that are encapsulated within VLPs thatcontain an immunogenic or active component and a stabilizer. Preferredstabilizers include sugar alcohols sorbitol and degraded gelatins. Theinvention further relates to stabilized vaccines for the treatment orprevention of infections cause by bacterial, viral, parasitic or otherinfections and the manufacture of such vaccines.

2. Description of the Background

HPV, or human papillomavirus, is a human DNA virus of the papillomavirusfamily of viruses. Similar to other papillomaviruses, HPVs infectskeratinocytes of the skin and mucous membranes. Typically, HPVinfections are subclinical and cause little to no physical symptoms.Infections can become clinical and lead to the development of benignpapilloma, also called warts or squamous cell papilloma. Clinicalinfections can also become cancerous developing into cancer of, forexample, the throat and reproductive tissues such as the cervix.

A large majority of clinical HPV infections regress to subclinical inone to two years. In patients where the subclinical infection persists,which occurs in five to ten percent of infected women, there is highrisk of developing precancerous lesions of the vulva and cervix thatprogress to invasive cancer. As progression from subclinical to clinicalinfection takes years, there are many opportunities for detection andtreatment of pre-cancerous lesions.

In developed countries, cervical screening using a Papanicolaou (Pap)test or liquid-based cytology is used to detect abnormal cells that maydevelop into cancer. If abnormal cells are found, women are invited tohave a colposcopy during which biopsies can be taken and abnormal areasremoved. The removal of abnormal cells is highly effective in preventingthe development of cervical cancer.

Two HPV vaccines are available, CERVARIX™ and GARDASIL™. Each preventsinfection caused by HPV types 16 or 18. These two strains are believedto be responsible for over seventy percent of cervical cancers. As withall vaccines, HPV vaccines need to be stabilized during transportationand storage and therefore contain stabilizers. Stabilizers are chemicalcompounds added to a vaccine formulation to enhance vaccine stabilityduring low temperature storage or storage post-lyophilization.

One such chemical stabilizer is referred to as SPGA and contains 218 mMsucrose, 3.76 mM KH₂PO₄, 7.1 mM K₂HPO₄, 4.9 mM potassium glutamate, and1% serum albumin. Modifications of SPGA include monosodium glutamate asa substitute for monopotassium glutamate, starch hydrosylate such asglucose or dextran as a substitute wholly or partly for sucrose, andcasein or PVP substituted wholly or partly for albumin. Anotherwell-known chemical stabilizer comprises approximately 3.5% hydrolyzedgelatin, 3.5% sorbitol, 1.0% Medium 199, along with minimal amounts ofsodium bicarbonate and phenol red. Other stabilizers comprise minuteamounts of DPG solution, which contains, among other compounds,cysteine, glutathionine, ascorbic acid, vitamin A and USP. Additionalprocesses and stabilizing agents are disclosed in U.S. Pat. Nos.3,985,615; 3,915,794; 4,000,256; 4,147,772; 4,537,769; 4,555,401;4,849,358; 4,985,244; 5,139,776; 7,998,488; 8,142,795; 8,551,523;8,557,253; 8,795,683 and International Application Publication No.WO1998028000, each of which is incorporated herein by reference.

Nevertheless, all of these stabilizers have shortcomings, either aninability to maintain stability in higher temperatures or fail after aperiod of time less than optimal. Thus, a need currently exists for aneffective stabilization chemical formulation and/or process thatmaintains immunogenicity of the vaccine and imparts no side effects.

SUMMARY OF THE INVENTION

In general, the invention is directed to compositions and methods forstabilizing vaccine formulation and especially human papilloma virus(HPV) vaccines.

One embodiment of the invention is directed to vaccines comprising VLPs(virus like particles) that encapsulate an immunogenic component and astabilizing agent. The immunogenic component comprises theimmune-stimulating portion or active portion of the vaccine thatgenerates an immune response. Preferred immune stimulating portionsinclude immunogenic portions of an infectious virus, bacterium orparasite. Preferred VLPs comprise structural components of a virus suchas, for example, the structural components of hepatitis virus or humanpapilloma virus (HPV), and VLP do not contain genetic material thatpermits replication of particles. Preferably the stabilizing agentcomprises one or more gelatins, one or more amino acids, or one or moresugar alcohols, or any combination thereof. Preferred gelatins aredegraded chemically or mechanically to fragments with an averagemolecular weight of ten kilodaltons or less, or eight kilodaltons orless. Also preferably, vaccines of the invention do not require theaddition of aluminum compounds such as aluminum phosphate. Vaccines ofthe invention may be maintained for long periods of time as a liquid, oreven longer periods of time as a lyophilized powder. Preferred vaccinesfurther contain pharmaceutically acceptable carriers such as, forexample, an alcohol, propylene glycol, fatty alcohols, triglycerides,fatty acid esters, mineral oils, liquid petrolatum, isopropylpalmitate,polyethylene ethanol, polyoxyethylene monolauriater, sodium laurylsulfate, an anti-oxidant, a humectant, a viscosity stabilizer ormodifier, a colorant or a flavoring agent.

Another embodiment of the invention is directed to methods for theadministration of stabilized vaccines of the invention to a patient.Preferred patients are mammals that have a functioning immune system.Methods comprise determining the therapeutically effective amount of thevaccine to be administered, and administering that amount of the vaccineto a patient in need thereof. Preferably the vaccine is a lyophilizedpowder and reconstituted to an aqueous or non-aqueous liquid prior toadministration to the patient. Also preferably the vaccine isadministered as a liquid and administering is intra-muscular,intra-peritoneal, or intra-venous. Preferred patients include, but arenot limited to an infant, a toddler, an adolescent, an adult or asenior. Preferably administration of a vaccine of the invention does notgenerate a general inflammation response in the patient or localinflammation at the site of administration.

Another embodiment of the invention is directed to methods for themanufacture of stabilized vaccines of the invention comprised of VLPs,an immunogenic agent, and a stabilizing agent, comprising: generatingVLP comprising structural components of human hepatitis virus; mixingthe components with an immunogenic agent and a stabilizing agent; andforming VLPs that encapsulate the immunogenic agent and the stabilizingagent. Preferably the structural components, the immunogenic agent andthe stabilizing agent are mixed at approximately equimolar amounts. Alsopreferably the VLPs that encapsulate the immunogenic agent and thestabilizing agent are in an aqueous mixture. Preferably manufacturingcomprising lyophilizing the aqueous mixture. Also preferably, noaluminum-containing compounds are added to the vaccine composition.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE INVENTION

There are many vaccines that are currently available that provideprotection against viral and bacterial infections. Each of thesevaccines contains various non-active components that increase stabilityand maintain the effectiveness of the vaccine over periods of time.

It has been surprisingly discovered that vaccine stability can beincreased by containing the vaccine in a virus-like particle (VLP).Preferably VLPs contain the immunogenic component of the vaccine plusone or more stabilizers and one or more pharmaceutically acceptablecarriers. Preferred stabilizers include sorbitol, amino acids, gelatindegraded to an average molecular weight of 10,000 MW or less, orcombinations thereof. Increased stability includes an increasedthermostability and an increased stability over time. Although theincreased stability herein is stated in reference to HPV vaccines, as isbelieved to be clear to those skilled in the art, the increasedstability is equally applicable to most any vaccine compositionincluding vaccines to prevent or treat viral, bacterial, parasiticand/or other infections.

Virus-like particles are comprised of viral structural proteins andresemble viruses, but are non-infectious and unable to replicate as theycontain no viral genetic material. The VLPs self-assemble fromexpression of the viral structural proteins, such as, for example,envelope or capsid proteins in vitro such as for example in a test tube,and/or in vivo such as for example in cell culture. VLPs of theinvention are preferably derived from hepatitis virus (e.g., Hepatitis Bvirus) and composed of the small HBV derived surface antigen (HBsAg),but may also be derived from the structural components of parvoviruses(e.g. adeno-associated virus), retroviruses (e.g. HIV), vesicularstomatitis virus (e.g., VSV), hepatitis virus (e.g. hepatitis A, B, C, Dor E virus), flaviviruses (e.g., West Nile virus, dengue virus,tick-borne encephalitis virus, yellow fever virus, Zika virus andseveral other viruses that cause encephalitis), and combinationsthereof. VLPs can be produced in a variety of cell culture systemsincluding mammalian cell lines, insect cell lines, yeast cell lines, andplant cell lines. VLPs may be formed in a buffered solution containingone or more of chelating and/or reducing agents.

Accordingly, one embodiment of the invention is directed to a vaccinecontaining an immunogenic agent and a stabilizing agent encapsulatedinto a VLP. The immunogenic agent of the VLP comprises an antigen orother structure that stimulates an immune response in an individual.Immunogenic agents include, but are not limited to peptides, proteins,lipids, fatty acids, polysaccharides, lipopolysaccharides. Typically,immunogenic agents are surface antigens of an infectious particle.Antigens may be specific for a particular infectious agent orcombination of agents such as, for example, viral infectious agents(e.g., enterovirus, hepatitis virus, human immunodeficiency virus {HIV},human papilloma virus {HPV}, influenza virus, pertussis virus, rubellavirus, tetanus, varicella virus {VZV}, flavivirus, West Nile virus,dengue virus, tick-borne encephalitis virus, yellow fever virus, Zikavirus), bacterial infectious agents (e.g., chlamydia, clostridium,diphtheria, meningococcal, streptococcal, staphylococcal, pneumococcal),parasitic infectious agents (e.g., giardia, malaria {plasmodium}),and/or an agent that causes sepsis or septicaemia. Preferably the immuneresponse is sufficient to protect the individual from subsequentinfections for a period of time. Preferably the vaccine is effective andprotects a patient from infection for six months or greater, one year orgreater, two years or greater, five years of greater, or ten years orgreater The immune response generated in response to the immunogenicagent of the invention generates a humoral immune response, a cellularimmune response, or preferably both in the individual. Preferredcellular response include a T cell response, and/or a phagocyticresponse, and also preferably a memory cell response.

Preferred stabilizing agents to include in VLPs of the inventioninclude, but are not limited to one or more of carbohydrates such asglucose and/or fructose, sugar alcohols such as glycerol, erythritol,threitol, avabitol, xylitol, ribitol, mannitol, galactitol, fucitol,iditol, inositol, volemitol, isomalt, lacitol, sorbitol, maltotriitol,maltotetraitol, polyglyitol or dextrin, dehydrated sugar alcohols suchas isosorbide, amino acids such as arginine, proline, and/or lysine,substituted amino acids such as hydroxyl-alanine, animal-derivedgelatins such as human collagen, BSA, and/or fish gelatin, and/orplant-derived carbohydrates such as pectin and cellulose. Preferablygelatins of the invention are degraded to an average molecular weight of100 kilodaltons (kD) or less, 50 kD or less, 20 kD or less, 10 kD orless, 8 kD or less, or 5 kD or less. Also preferably, vaccines of theinvention do not require the addition of aluminum, magnesium ormanganese compounds such as aluminum phosphate or other forms ofaluminum, magnesium or manganese, or preferably any metal compound.Stabilizing agents may be added at from 1-10% of the dry weight of theVLP components, or from 1-10% of the dry weight of the VLP componentsplus the immunogenic agent, or preferably from 2-4% of the dry weight ofeither composition. Also preferably, the stabilizing agent is at aconcentration of from 1 mM to 50 mM of the aqueous mixture, preferablyat a concentration of from 10-20 mM.

Preferably the vaccine is maintained as a liquid, but the liquid may belyophilized and stored as a dry powder until use wherein the powder isrehydrated in an appropriate carrying agent (aqueous or non-aqueous) foradministration to the individual. Administration is preferable byinjection which may be intra peritoneal (i.p.), intra muscular (i.m.),and/or intra venous (i.v.), or localized to the site of an infection.

Preferably the vaccines of the invention as liquids or powders arestable at 4° C. or greater, 15° C. or greater, 25° C. or greater, 37° C.or greater, 40° C. or greater, 50° C. or greater, or 100° C. or greater.Also preferably, the vaccines of the invention as liquids or powers arestable at 15° C. or less, more preferably to 0° C. or less, morepreferably to minus 20° C. or less, and more preferably to minus 50° C.or less. Also preferably, stability of the vaccines is maintained forsix month or greater, for eight months or greater or for twelve monthsor greater. Preferably the vaccines of the invention are stable throughvarying temperatures over time which may include multiple freezing andthawing.

Another embodiment of the invention is directed to methods for theadministration of vaccines of the invention to patients in need thereoffor treating or preventing an infection. The method comprisesadministering a therapeutically effective amount of the vaccine of theinvention to a mammal, comprising determining the therapeuticallyeffective amount of the vaccine to be administered. The therapeuticallyeffective amount is typically determined by based on the weight of themammal and the strength or responsiveness of the patient's immune systemand can be determined by those skilled in the art. The therapeuticallyeffective amount is administered to a patient in need thereof, which maybe to treat an active or suspected infection or prevent an infection.The vaccine may have been obtained from a lyophilized powder andreconstituted to an aqueous or non-aqueous liquid prior toadministration to the patient. Preferably the vaccine is administered asa liquid, which may be intra-muscular, intra-peritoneal, orintra-venous, and the patient may be an infant, a toddler, anadolescent, an adult or a senior. Surprisingly, the vaccine of theinvention does not generate side effects such as redness or inflammationat the injection site, and does not generate a generalized fever orinflammation, or other unwanted side effects for the patient. Preferablyan immunologically effective vaccine contains only the fully formed VLPscontaining immunogenic and stabilizing agents, and nothing further suchas, for example, no added ionic or non-ionic surfactants.

Another embodiment of the invention is directed to method for themanufacture of vaccines of the invention. Structural components ofviruses are obtained by methods well known to those skilled in the artand exclusive of any nucleic acid material that would allow thecomponents and resulting particles to replicate. Predetermined molaramounts of the structural components are mixed, preferably at roomtemperature or below, with approximately equivalent molar amounts of oneor more immunogenic agents and one or more stabilizing agents of theinvention, such that the VLPs encapsulate the one or more immunogenicagents and the one or more stabilizing agents in roughly equivalentamounts. The fully formed VLPs are separated from unformed VLPs and freestructural and other materials preferably by filtration, centrifugationor another method known to those skilled in the art, thereby creatingfully formed VLPs containing one more immunogens and one or morestabilizing agents. The fully formed VLPs may be stored as an aqueous(e.g., water or saline) or non-aqueous (e.g., oils, fatty acids)mixture, or lyophilized and stored as a powder. Preferably storage untiluse is without significant loss of immunogenic activity and, forexample, may be for one month or longer, four months or longer, sixmonths or longer, or more preferably one year or longer and at ambienttemperatures, whether as a liquid or a powder. Storage of vaccinewithout loss of immunogenic activity may also be at less than ambienttemperature such as, for example, at 20° C. or less, at 10° C. or less,at 4° C. or less, or at 0° C. or less.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications, are specifically and entirely incorporated by reference.The term comprising, where ever used, is intended to include the termsconsisting and consisting essentially of. Furthermore, the termscomprising, including, and containing are not intended to be limiting.It is intended that the specification and examples be consideredexemplary only with the true scope and spirit of the invention indicatedby the following claims.

The invention claimed is:
 1. A method of treating or preventing aninfection comprising: determining a therapeutically effective amount ofa composition that has been maintained as a liquid to be administered toa mammal in need thereof, wherein: the composition comprises virus-likeparticles (VLPs), an immunogenic component that is protective againstthe infection and a stabilizing agent, the VLP comprises the structuralcomponents of hepatitis virus, the immunogenic component comprises acomponent of human papilloma virus, and the stabilizing agent comprisesa degraded gelatin with an average molecular weight of ten kilodaltonsor less, and the composition has not been maintained in a non-liquidstate; and administering the therapeutically effective amount of thecomposition to the mammal.
 2. The method of claim 1, whereinadministering is intra-muscular, intra-peritoneal, or intra-venous. 3.The method of claim 1, wherein the patient is an infant, a toddler, anadolescent, an adult or a senior.
 4. The method of claim 1, whereinadministration does not generate a general inflammatory response orlocal inflammation at the site of administration.
 5. The method of claim1, wherein the stabilizing agent comprises one or more amino acids, orone or more sugar alcohols, or any combination thereof.
 6. The method ofclaim 1, wherein the degraded gelatin is degraded chemically ormechanically.
 7. The method of claim 1, wherein the degraded gelatin isdegraded to an average molecular weight of eight kilodaltons or less. 8.The method of claim 1, wherein the stabilizing agent further comprises asugar alcohol.
 9. The method of claim 8, wherein the sugar alcoholcomprises sorbitol.
 10. The method of claim 1, wherein the compositiondoes not contain an aluminum compound.
 11. The method of claim 1,wherein the composition further contains a pharmaceutically acceptablecarrier selected from the group consisting of alcohol, propylene glycol,fatty alcohols, triglycerides, fatty acid esters, mineral oils, liquidpetrolatum, isopropylpalmitate, polyethylene ethanol, polyoxyethylenemonolauriater, sodium lauryl sulfate, an anti-oxidant, a humectant, aviscosity stabilizer or modifier, a colorant or a flavoring agent.
 12. Amethod of generating an immune response comprising: preparing animmunogenic composition that has been maintained as a liquid comprisingVLPs, an immunogenic component, and a stabilizing agent, wherein the VLPcomprises the structural components of hepatitis virus, the immunogeniccomponent comprises a component of human papilloma virus, and thestabilizing agent comprises a degraded gelatin with an average molecularweight of ten kilodaltons or less, and wherein the immunogeniccomposition has not been maintained in a non-liquid state; administeringthe immunogenic composition to a mammal; and generating an immuneresponse in the mammal.
 13. The method of claim 12, wherein the mammalis an infant, a toddler, an adolescent, an adult or a senior.
 14. Themethod of claim 12, wherein administration does not generate a generalinflammatory response or local inflammation at the site ofadministration.
 15. The method of claim 12, wherein the stabilizingagent further comprises one or more amino acids, or one or more sugaralcohols, or any combination thereof.
 16. The method of claim 12,wherein the degraded gelatin is degraded to an average molecular weightof eight kilodaltons or less.
 17. The method of claim 12, wherein theimmunogenic composition does not contain an aluminum compound.
 18. Amethod of generating an immune response against an infectious agentcomprising: preparing an immunogenic composition as a liquid comprisingVLPs, a stabilizing agent, and an immunogenic component, wherein the VLPcomprises the structural components of hepatitis virus, the stabilizingagent comprises a degraded gelatin, the immunogenic component comprisesa surface antigen of human papilloma virus, and the immunogeniccomposition does not contain an aluminum compound, and wherein theimmunogenic composition has not been maintained in a non-liquid state;administering the liquid immunogenic composition to a mammal, whereinthe administration does not generate a general inflammatory response orlocal inflammation at the site of administration; and generating animmune response against the infectious agent in the mammal.
 19. Themethod of claim 18, wherein the degraded gelatin is degraded chemicallyor mechanically.
 20. The method of claim 18, wherein the degradedgelatin is degraded to an average molecular weight of eight kilodaltonsor less.
 21. The method of claim 18, wherein the degraded gelatin isdegraded to an average molecular weight of five kilodaltons or less. 22.The method of claim 1, wherein the composition is stable at temperaturesfrom 4° C. to 50° C.
 23. The method of claim 1, wherein the compositionis stable through freezing and thawing.
 24. The method of claim 12,wherein the immunogenic composition is stable at temperatures from 4° C.to 50° C.
 25. The method of claim 12, wherein the immunogeniccomposition is stable through freezing and thawing.
 26. The method ofclaim 18, wherein the immunogenic composition is stable at temperaturesfrom 4° C. to 50° C.
 27. The method of claim 18, wherein the immunogeniccomposition is stable through freezing and thawing.